Existing methods for, the chemical synthesis of proteins include stepwise solid phase synthesis, and fragment condensation either in solution or on solid phase. The classic stepwise solid phase synthesis of Merrifield involves covalently linking an amino acid corresponding to the carboxy-terminal amino acid of the desired peptide chain to a solid support and extending the polypeptide chain toward the amino end by stepwise coupling of activated amino acid derivatives having activated carboxyl groups. After completion of the assembly of the fully protected solid phase bound peptide chain, the peptide-solid phase covalent attachment is cleaved by suitable chemistry and the protecting groups removed to give the product polypeptide.
Some disadvantages of the stepwise solid phase synthesis method include: incomplete reaction at the coupling and deprotection steps in each cycle results in formation of solid-phase bound by products. Similarly, side reactions due to imperfections in the chemistry, and or impurities present in the reagents/protected amino acids, all lead to a multiplicity of solid phase bound products at each step of the chain assembly and to the formation of complex product mixtures in the final product. Thus, the longer the peptide chain, the more challenging it is to obtain high-purity well-defined products. Due to the production of complex mixtures, the stepwise solid phase synthesis approach has size limitations. In general, well-defined polypeptides of 100 amino acid residues or more are not routinely prepared via stepwise solid phase synthesis. Synthesis of proteins and large polypeptides by this route is a time-consuming and laborious task.
The solid phase fragment condensation approach (also known as segment condensation) was designed to overcome the difficulties in obtaining long polypeptides via the solid phase stepwise synthesis method. The segment condensation method involves preparation of several peptide segments by the solid phase stepwise method, followed by cleavage from the solid phase and purification of these maximally protected segments. The protected segments are condensed one-by-one to the first segment, which is bound to the solid phase.
Often, technical difficulties are encountered in many of the steps of solid phase segment condensation. See E. Atherton, et al., “Solid Phase Fragment Condensation—The Problems,” in Innovation and Perspectives in Solid Phase Synthesis 11-25 (R. Epton, et al. 1990). For example, the use of protecting groups on segments to block undesired ligating reactions can frequently render the protected segments sparingly soluble, interfering in efficient activation of the carboxyl group. Limited solubility of protected segments also can interfere with purification of protected segments. See K. Akaji et al., Chem. Pharm. Bull.(Tokyo) 33:184-102 (1985). Protected segments are difficult to characterize with respect to purity, covalent structure, and are not amenable to high resolution analytical ESMS (electrospray mass spectrometry) (based on charge). Racemization of the C-terminal residue of each activated peptide segment is also a problem, except if ligating is performed at Glycine residues. Moreover, cleavage of the fully assembled, solid-phase bound polypeptide from the solid phase and removal of the protecting groups frequently can require harsh chemical procedures and long reaction times that result in degradation of the fully assembled polypeptide.
Segment condensation can be done in solution rather than on solid phase. See H. Muramatsu et al., Biochem. and Biophys. Res. Commn. 203(2):1131-1139 (1994). However, segment condensation in solution requires purification of segments prior to ligation as well as use of protecting groups on a range of different side chain functional groups to prevent multiple undesired side reactions. Moreover, the ligation in solution does not permit easy purification and wash steps afforded by solid phase ligations. Furthermore, the limitations with respect to solubility of protected peptide segments and protected peptide intermediate reaction products are exacerbated.
Chemical ligating of minimally protected peptide segments has been explored in order to overcome the solubility problems frequently encountered with maximally protected peptide segments. See Cheng, et al., Chemical Synthesis of Human θ-endorphin(1-27) Analogs by Peptide Segment Coupling. Int. J. Pept. Protein Res. 38:70-78 (1991); J. Blake, Total Synthesis of S-Carbamoylmethyl Bovine Apocytochrome c by Segment Coupling, Int. J. Pept. Protein Res. 27:191-200 (1986); and H. Hojo et al., Protein Synthesis using S-Alkyl Thioester of Partially Protected Peptide Segments, Synthesis of DNA-Binding Protein of Bacillus stearothermophilus, Bull. Chem. Soc. Jpn. 65:3055-3063 (1992). However, this method still requires the use of protecting groups on all Lysine side chain amino groups, selective N-α protection of one or more segments, and laborious purification steps, involving purification, reprotection, and repurification.
The use of multiply protected peptide segments is incompatible with the overall scheme of engineering proteins using peptides produced by means of recombinant DNA expression as a source. Protected peptide segment methods are labor-intensive, and the protected peptide segments have unpredictable handling properties, partly due to the solubility and ligating difficulties of protected peptide segments. Often, large protected peptide segments are minimally soluble in even the most powerful polar aprotic solvents such as dimethylsulfoxide (DMSO) and dimethylformamide (DMF). The problem of insolubility in protected peptide segments has been addressed with limited success in several ways, including the use of (1) partial protecting group strategy which masks all side chains except those of Ser, Thr, and Tyr; (2) minimal protecting group strategy that masks only thiol and amino side chains; and (3) using reversible protection of a backbone amide moiety to prevent aggregation/insolubility. Protecting groups used in the latter approach alter peptide conformations. Use of backbone protecting groups is not yet straightforward or predictable and requires significant experimentation for each target polypeptide chain.
There are a number of techniques for ligating unprotected peptide segments via unnatural backbone linkages. In contrast, there are few methods for achieving a “native chemical ligation.” A “native chemical ligation” is the chemoselective reaction of unprotected or N-terminal Cysteine protected peptide segments with another unprotected peptide segment resulting in the formation of a ligated peptide with an amide bond at the ligation site. The fully assembled target polypeptides of the invention comprise one, two or more native chemical ligation sites.
Accordingly, there is a need in the art for rapid methods of synthesizing assembled polypeptides via chemical ligation of two or more unprotected peptide segments using a solid support, with improved yields and facilitated handling of intermediate products.
The present invention makes possible, inter alia, the rapid solid-phase synthesis of large polypeptides with a natural peptide backbone via native chemical ligation of two or more unprotected peptide segments where none of the reactive functionalities on the peptide segments need to be temporarily masked by a protecting group. The present invention accomplishes for the first time, solid phase sequential chemical ligation of peptide segments in an N-terminus to C-terminus direction, with the first solid phase-bound unprotected peptide segment bearing a C-terminal α-thioester that reacts with another unprotected peptide segment containing an N-terminal Cysteine and a C-terminal thioacid.
Other embodiments of the invention also permit solid-phase native chemical ligation in the C- to N-terminus direction, with temporary protection of N-terminal cysteine residues on an incoming (second) peptide segment. Those of ordinary skill in the art will readily appreciate that the invention may also include the use of nonnative chemical ligation to sequentially ligate peptide segments via unnatural linkages on a solid phase. Alternatively, the invention may include the use of native chemical ligation of peptide segments wherein said peptide segments comprise one or more unnatural backbone linkages.